An efficient transient gene expression system for protein subcellular localization assay and genome editing in citrus protoplasts

Protoplast has been widely used in biotechnologies to circumvent the breeding obstacles in citrus, including long juvenility, polyembryony, and male/female sterility. The protoplast-based transient gene expression system is a powerful tool for gene functional characterization and CRISPR/Cas9 genome editing in higher plants, but it has not been widely used in citrus. In this study, the polyethylene glycol(PEG)-mediated method was optimized for citrus callus protoplast transfection, with an improved transfection efficiency of 68.4%. Consequently, the efficiency of protein subcellular localization assay was increased to 65.8%, through transient expression of the target gene in protoplasts that stably express the fluorescent organelle marker protein. The gene editing frequencies in citrus callus protoplasts reached 14.2% after transient expression of CRISPR/Cas9 constructs. We demonstrated that the intronic polycistronic tRNAgRNA(inPTG) genome editing construct was functional in both the protoplast transient expression system and epicotyl stable transformation system in citrus. With this optimized protoplast transient expression system, we improved the efficiency of protein subcellular localization assay and developed the genome editing system in callus protoplasts, which provides an approach for prompt test of CRISPR vectors.

Efficient gusA Transient Expression in Porphyra yezoensis Protoplasts Mediated by Endogenous Beta-tubulin Flanking Sequences

Endogenous tubulin promoter has been widely used for expressing foreign genes in green algae, but the efficiency and feasibility of endogenous tubulin promoter in the economically important Porphyra yezoensis (Rhodophyta) are unknown. In this study, the flanking sequences of beta-tubulin gene from P. yezoensis were amplified and two transient expression vectors were con-structed to determine their transcription promoting feasibility for foreign gene gusA. The testing vector pATubGUS was constructed by inserting 5’- and 3’-flanking regions (Tub5’ and Tub3’) up- and down-stream of β-glucuronidase (GUS) gene (gusA), respectively, into pA, a derivative of pCAT?3-enhancer vector. The control construct, pAGUSTub3, contains only gusA and Tub3’. These con-structs were electroporated into P. yezoensis protoplasts and the GUS activities were quantitatively analyzed by spectrometry. The results demonstrated that gusA gene was efficiently expressed in P. yezoensis protoplasts under the regulation of 5’-flanking sequence of the beta-tubulin gene. More interestingly, the pATubGUS produced stronger GUS activity in P. yezoensis protoplasts when com-pared to the result from pBI221, in which the gusA gene was directed by a constitutive CaMV 35 S promoter. The data suggest that the integration of P. yezoensis protoplast and its endogenous beta-tubulin flanking sequences is a potential novel system for foreign gene expression.

The protoplasts isolation,culture and shoots regeneration of broccoli(Brassica oleracea var.italica)

Eight F1-hybrid cultivars of broccoli were studied.We obtained cell division,celled colonies and p-calli in 5 cultivars,roots and shoots regeneration in one cultivar.The leavesof propagated plantlets in vitro were cut into 1—2mm pieces,isolated with an enzyme solutioncontaining 2% cellulase and 1%macerase on a rotary shaker（50 rpm,21℃,3h,2500 lux light）,and purified with a 0.5M sucrose solution.The purified protoplasts were placed on a drop of 1%agarose.2—3 ml liquid medium was added around the agarose drops,and all of the cultures wereincubated at 25℃ under light（4000 lux）for 16 hours.3—5 days after isolation the cell divisionwas found.About 7 days after incubation 4 multicellular colonies were formed.After 3—5 wksome p-calli were developed.When the p-calli were 2—3 mm in diameter it was transferred to asolidified medium.Once they were developed to 1 cm in diameter they were transferred on a re-generation medium.About 5 months after incubation some roots and shoots grown from the calliwere

Factors affecting the production and regeneration of protoplasts: the case of the mycorrhizal fungus Tulasnella calospora from roots of orchids

Protoplast isolation is relevant for many different applications and has been principally used in proceduresnvolving genetic manipulation. In this study, the age of mycelium, osmotic stabilizers, enzyme, incubation temperature and incubation time were evaluated in terms of their effects on protoplast yield. The young mycelia （3 d） of Tulasnella calospora were digested for 6 h at 30℃ in a mixture of 1.2 mol·L-1 MgSO_4 ＋ 10 mmoI·L-1 K2HPO4 as the osmotic stabilizer, with a 1.0% lysing enzyme and 1.5% driselase： more than 106 protoplasts mL-1 were obtained. When collected 3y density gradient centrifugation, the concentration of protoplasts can reach 107-108 protoplasts mL-1, an amount suitable enough for experiments of transformation in fungi. For every 10_5 protoplasts, about 15-25 protoplasts can egenerate after 24-36 h cultivation in a liquid medium and after 8-10 d in an agar medium. This study produced an efficient method for protoplast production, reverting them into a typical mycelia morphology using a Tulasnella calospora solate.

Plant regeneration of protoplasts isolated from suspension cells derived from leaf blale of Oryza sative

We used the leaf blade of rice (cultivars were Nonghu 6, Sugeng 2, Huyou 2 and Hanfeng) as initial material for protoplast culture, and a great number of regenerated plants were obtained. Rice seeds were sterilized and germinated. The immature leaves were cut into 3-5 mm pieces when the third or forth leaf appeared. Leaf pieces were inoculated on MS medium with 2,4-D 4 mg/1, NAA 2mg/1 and IAA Img/1. After 2 wk culture, calli were induced and subcultured once or twice for multiplication. 3-5 g calli were transferred to the modified MS liquid medium with 2,4-D 2 mg/1 and KT 0.5mg/1 for suspension culture. Embryogenic cell suspension was established after 2 mo culture. The effect of the growth period of suspension cells on the

STUDIES ON COLOR TYPE VARIANTSFROM MUTAGENIZED PROTOPLASTS OFPORPHYRA HAITANENSIS CHANG ET ZHENG& P. YEZOENSIS UEDA (RHODOPHYCEASE )

Isolated protoplasts from thalli of Porphyra haitanensis and Porphyra yezoensis were treated with colchicine or irradiated by ultraviolet (UV ). Several types of color variants were observed among the protoplast offspring. After treatment with colchicine: (1) 0.04-0.09% of red type variants in P. haitanensis were obtained; (2) The rate of red type variants and the variegated chimeral thalli composed of red type and wild type of sectors were 6.31- 1.11% in P. yezoensis. After irradiation with UV: (1) 3.5- 10.5% of red type variants in P. yezoensis were obtained: (2) 0.5-2-0% of red type variants and the variegated chimeral thalli composed of red type and wild type of sectors were obtained in P. haitanensis. Colchicine and UV’s mutangenic effects on P. yezoensis protoplasts were stronger than those on P. haitanensis protoplasts. The most efficient concentration of colchicine was 0.05%. The optimal length of UV-radiation was 1/2 min (radiation distance 5 cm). The red type variants induced, by colchicine

Optimization of Isolation and Culture of Protoplasts in Alfalfa (<i>Medicago sativa</i>) Cultivar Regen-SY

Alfalfa (Medicago sativa) is an important forage crop belonging to the Fabaceae family. It is cultivated across the world for fodder and originated in Asia. Alfalfa cultivar Regen-SY was used in this study which is a hybrid of first-generation self-parents from Regen-S (M. sativa) and Regen-Y (Medicago falcata) research cultivars. The main objective of the study was to optimize conditions for the isolation and liquid culture of alfalfa Regen-SY protoplasts. Several factors like enzyme combination, incubation time, plant age, centrifugation speed and shaker speed affecting protoplast isolation and culture were optimized in the study. The yield and viability of the protoplasts was determined by using hemocytometer and Fluorescein diacetate (FDA) staining respectively. Results showed that factors like enzyme combination, incubation time, plant age, centrifugation speed and Mannitol concentration significantly (p ≤ 0.05) affect protoplast yield and viability whereas shaker speed didn’t result in any significant difference in the yield and viability of protoplasts. Using optimum conditions protoplasts were cultured in the liquid medium and microcalli formation was achieved after five weeks of the culture. The protocol established in this study will assist researchers in the isolation and culture of protoplasts in alfalfa and will accelerate the research processes like protoplast fusion and genetic engineering.

Studies on the isolation and culture of protoplasts from Kappaphycus alvarezii

In this study, protoplasts were successfully isolated from Kappaphycus alvarezii using snail enzymes, abalone enzymes and cellulase. The optimum enzymic ratio was fixed to be 20% of abalone enzyme, 12% of cellulase and the osmotic stabilizer was 2.0 mol/L glucose. The optimum enzymic hydrolysis conditions were found to be dark enzymolysis at 30°C continuing for 4.0 h. The resultant density and yield of protoplasts achieved 32.60×10^4 mL-1, 65.20×10^4 g-1 tissue for Kappaphycus alvarezii. Finally, under the temperature of 20°C, light intensity of 1 500–2 000 lx and photoperiod of 12 h/d, two developmental pathways were investigated：（1） callus-like cell mass and regenerated plantlet occurred on protoplast;（2） young shoots and calluslike cell mass occurred in tissue blocks after enzymolysis.

Plant Regeneration from Protoplasts of Wide Compatible Rice

Rice selection 02428 and T984(Oryzasativa L.ssp.japonica)were germplasmresources with wide compatibility.Mature embryos of rice cultured on Lin-smaier and Skoog medium with 2.5 mg/l2,4-D,1.0 mg/l thiamine-HCL,3%(W/V)sucrose and 0.7%(W/V)agar,pH 5.8(LS2.5)were used for callus initiation.Cultures were

Isolation and callus formation of Gracilariopsis bailiniae(Gracilariales, Rhodophyta) protoplasts

This paper reports the first successful isolation of protoplasts from G racilariopsis bailiniae and their callus formation. The base solution type, concentration of isolating enzymes, concentration of sorbitol, incubation time, temperature and pH of the enzyme solution were tested to optimize the protoplast yield. The optimized isolation conditions were: 40% base solution 3(deionized water containing 25 mmol/L MESTris and 25 mmol/L CaCl 2 ·2 H 2 O) and 60% crude Marinomonas sp. YS-70 agarase solution, containing 2% w/v cellulase, 1% w/v macerozyme R-10 and 0.4 mol/L sorbitol, with incubation for 4 h at 28°C and pH 6.5. The highest yield of viable protoplasts, which was obtained in these conditions, was(1.75±0.25)×10 6 cells/g fresh weight. Cell wall regeneration of most protoplasts from G. bailiniae was complete within 60 h and the first division of cells happened after ≥3 days. Two division types were observed in the first division of protoplasts from G. bailiniae— asymmetric division and symmetric division. After the first division, the cells underwent a series of divisions to form callus cell masses.

Transformation of rice protoplasts

Protoplasts of a wide-compatibility variety of rice(02428)were used as recipients for transformation offoreign DNA sequences.Direct gene transfer was proceeded under the existence of polyethylene glycol(PEG).In situ histochemical assay showed the activity of beta-glucuronidase,whose gene has been trans-

PEG Delivery of a Wheat Storage Protein Gene into Rice Protoplasts

An effiecent cell suspension culture system has been initiated using callus obtained frommature seeds of the Chinese Japonica variety Eryi 105 as a source.Over the last 12 months,using the'Nottingham Method',protoplasts have been produced from these cell suspen-sions,and via the PEG method of transformation.45 regenerated plants were obtained fromDNA-treated protoplasts selected by 80μg/ml G418.The research of molecular analysis onthose plantlets is being carried on by Plant Genetic Manipulation Group,Department of LifeScience,University of Nottingham,U.K.

Rice Protoplasts Isolated Directly from Mature-Embryo-Derived Calli

Mature-embryo-derived calli or japonica rice (Oryza sativa L)Taipei 309 were used for replicated protoplast isolation experiments. Six out of nine callus lines produced protoplasts with satisractory yield of 5. 20×106～8. 96× 106 protoplasts/g FW(fresh weight).The remaining three callus lines Initiated from seeds of cryopreserved- callus-derived plants had rooty calli, resulting in low yield or protoplasts and a large number or isolated banana- shape intact cells. Viability or protoplasts ranged 87.46% ～94.15%.The average size or Protoplasts was 207. 49～379. 04μm2 in different callus lines.Comparitive experiments were also carried out using both calli and suspension culture cells for protoplast isolation. The results demonstrated that protoplast isolation of calli was a substantially simplified and reliable method for preparing rice protoplasts.

Formation and Growth of Bryopsis hypnoides Lamouroux Regenerated from Its Protoplasts

Abstract: Tissue culture, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and spectra analysis were used for studying the aggregation mechanism of protoplasts from Bryopsis hypnoides Lamouroux and the discrepancy between the protoplast-regenerated plants and the wild type. The aggregation of protoplasts from B. hypnoides was observed in natural seawater and artificial seawater with different pH values, and the location and mechanism of the materials causing the aggregation were also studied. Results showed that the protoplasts could aggregate into some viable spheres in natural seawater and subsequently grow into mature individuals. Aggregation of the protoplasts depended exclusively upon the pH value (6–11), and the protoplasts aggregated best at pH 8–9. Some of the extruded protoplasts were separated into two parts by centrifugation: the pellet (PO) and the supernatant (PL). The PO could aggregate in artificial seawater (pH 8.3) but not in PL. No aggregation was found in PO cultured in natural seawater containing nigericin, which can dissipate the proton gradients across the membrane. These experiments suggest that the aggregation of protoplasts is proton-gradient dependent and the materials causing the aggregation were not in the vacuolar sap, but located on the surface or inside the organelles. Furthermore, the transfer of the materials across the membrane was similar to z\pH-based translocation (δpH/TAT) pathway that occurs in the chloroplasts of higher plants and bacteria. Obvious discrepancies in both the total soluble proteins and the ratio of chlorophyll a to chlorophyll b between the regenerated B. hypnoides and the wild type were found, which may be related to the exchange of genetic material during aggregation of the organelles. In the process of development, diatom Amphora coffeaeformis Agardh attached to the protoplast aggregations, retarding their further development, and once they were removed, the aggregations immediately germinated, which showed that diatoms can affect the development of other algae.

Dissection of miRNA Pathways Using Arabidopsis Mesophyll Protoplasts

MicroRNAs （miRNAs） control gene expression mostly post-transcriptionally by guiding transcript cleavage and/or translational repression of complementary mRNA targets, thereby regulating developmental processes and stress responses. Despite the remarkable expansion of the field, the mechanisms underlying miRNA activity are not fully understood. In this article, we describe a transient expression system in Arabidopsis mesophyll protoplasts, which is highly amenable for the dissection of miRNA pathways. We show that by transiently overexpressing primary miRNAs and target mimics, we can manipulate miRNA levels and consequently impact on their targets. Furthermore, we developed a set of luciferase-based sensors for quantifying miRNA activity that respond specifically to both endogenous and overexpressed miRNAs and target mimics. We demonstrate that these miRNA sensors can be used to test the impact of putative components of the miRNA pathway on miRNA activity, as well as the impact of specific mutations, by either overexpression or the use of protoplasts from the corresponding mutants. We further show that our miRNA sensors can be used for investigating the effect of chemicals on miRNA activity. Our cell-based transient expression system is fast and easy to set up, and generates quantitative results, being a powerful tool for assaying miRNA activity in vivo.

Dynamic Actin Controls Polarity Induction de novo in Protoplasts

Cell polarity and axes are central for plant morphogenesis. To study how polarity and axes are induced de novo, we investigated protoplasts of tobacco Nicotiana tabacum cv. BY-2 expressing fluorescently- tagged cytoskeletal markers. We standardized the system to such a degree that we were able to generate quantitative data on the temporal patterns of regeneration stages. The synthesis of a new cell wall marks the transition to the first stage of regeneration, and proceeds after a long preparatory phase within a few minutes. During this preparatory phase, the nucleus migrates actively, and cytoplasmic strands remodel vigorously. We probed this system for the effect of anti-cytoskeletal compounds, inducible bundling of actin, RGD-peptides, and temperature. Suppression of actin dynamics at an early stage leads to aberrant tripolar cells, whereas suppression of microtubule dynamics produces aberrant sausage- like cells with asymmetric cell walls. We integrated these data into a model, where the microtubular cytoskeleton conveys positional information between the nucleus and the membrane controlling the release or activation of components required for cell wall synthesis. Cell wall formation is followed by the induction of a new cell pole requiring dynamic actin filaments, and the new cell axis is manifested as elongation growth perpendicular to the orientation of the aligned cortical microtubules.

EFFECT OF T-2 TOXIN ON WHEAT LEAF PROTOPLASTS

Ⅰ. INTRODUCTIONCooking was the first who succeeded in getting a large number of protoplasts by using the enzymatic method in the 1960s. Now protoplasts have become an important tool in the research of plant physiology, plant genetics, phytopathology and membrane physiology. In the research of inter-relationship between protoplasts and pathotoxin,

Induction of Apoptosis in Protoplasts and Suspension Cultures of Plant Cells

Many studies have showed that apoptosis exists in plants. Our study shows that (1) menadione(VK3) induces apoptosis in suspension cultures of carrot cells; (2) heat shock induces apoptosis in suspension cultures of tobacco cells; and (3) ethrel induces apoptosis in carrot protoplasts. Some important indications of apoptosis were observed, including DNA laddering, TUNEL-positive reaction, condensation and degradation of nuclei.[

Cell size: a key determinant of meristematic potential in plant protoplasts

Metabolic pathway reconstruction and gene edits for native natural product synthesis in single plant cells are considered to be less complicated when compared to the production of non-native metabolites.Being an efficient eukaryotic system,plants encompass suitable post-translational modifications.However,slow cell division rate and heterogeneous nature is an impediment for consistent product retrieval from plant cells.Plant cell synchrony can be attained in cultures developed in vitro.Isolated plant protoplasts capable of division,can potentially enhance the unimpaired yield of target bioactives,similar to microbes and unicellular eukaryotes.Evidence from yeast experiments suggests that‘critical cell size’and division rates for enhancement machinery,primarily depend on culture conditions and nutrient availability.The cell size control mechanisms in Arabidopsis shoot apical meristem is analogous to yeast notably,fission yeast.If protoplasts isolated from plants are subjected to cell size studies and cell cycle progression in culture,it will answer the underlying molecular mechanisms such as,unicellular to multicellular transition states,longevity,senescence,‘cell-size resetting’during organogenesis,and adaptation to external cues.

Determination of rare earth elements in plant protoplasts by MAA

A preliminary study on the speciation of rare earth elements in plant cells has been carried out by molecular activation analysis (MAA). Mesophyll protoplasts of Brassica napus were isolated by enzymatic digestion. After being washed with isosmotic solution containing EDTA for several times, the protoplasts were purified by gradient centrifugation. Then the concentration of rare earth elements (REEs) in the protoplasts was determined by neutron activation analysis. The result shows that REEs can enter the cells of the plant.